Our long-term goal is to elucidate the mechanisms of splenic toxicity resulting from exposure to aromatic amines, and to develop preventive/therapeutic strategies. We use the industrial chemical aniline as a prototypic amine. Besides causing methemoglobinemia, hemolysis and hemolytic anemia, aniline exposure also results in selective splenic toxicity. The mechanism(s) for this selective splenic damage are not well understood. Studies in our previous funding period demonstrated that aniline exposure increased iron content, lipid peroxidation, protein oxidation and lipid-derived aldehyde-protein adducts (markers of oxidative stress) in rat spleens. These events are accompanied by such morphological changes as vascular congestion, increased sinusoids and fibroblasts, erythrophagocytocysis, capsular thickening and fibrosis. We hypothesize that aniline toxicity is caused by oxidative stress, and postulate the following sequence of events leading to splenic toxicity: Aniline (and/or its metabolites) damage erythrocytes, which are deposited in the spleen, and so increase total and reactive iron (free iron). This iron overload generates oxidants (probably hydroxyl radicals), causing oxidative stress leading to cellular dysfunction and fibrosis [increased collagen synthesis mediated by fibrogenic cytokines and activation of transcription factors)]. This hypothesized sequence of events will be tested by pursuing three specific aims: Aim 1 identify the oxidizing species and iron species that mediate aniline toxicity. Aim 2 will characterize the oxidized biomolecules in the spleens of aniline-exposed rats, especially the lipid peroxidation products and their protein and DNA adducts; the oxidized proteins will also be characterized. Aim 3 will examine how the induction of fibrogenic cytokines; activation of stress-induced transcription factor(s), and collagen synthesis contribute to aniline toxicity. Our project will elucidate the mechanism(s) of aniline-induced splenic toxicity, and will define the roles of oxidative stress, aldehyde products of lipid peroxidation, fibrogenic cytokines and transcription factor(s) in this process. Understanding the mechanism(s) of aniline toxicity will be very important in developing novel preventive and therapeutic strategies for aromatic amines, in general.